A Novel Fish Scale Derived Scaffold for Ocular Reconstruction

Abstract

 

Purpose

 

The purpose of this study was to determine (1) the biocompatibility of a decellularized fish scale derived scaffold with human corneal epithelial cells (CECs) in vitro, and (2) the optimal conditions for culturing primary CECs on the surface of the scaffold.

 

Methods

 

First, Scanning Electron Microscopy (SEM) was performed to determine the ultra structure of the scaffold. Next, primary CECs were cultured on the scaffolds in CnT-20 medium. Cultures were evaluated by light microscopy and immunofluorescence microscopy using antibodies against CK3/12, CK14, p63, FLT4 and ABCG2. To determine optimal culture conditions, primary CECs were cultured on scaffolds using different culture protocols: (A) Supplemental Hormonal Epithelial Medium (SHEM)+10% Foetal Bovine Serum (FBS) for 2 weeks, (B) CnT-20 medium for 2 weeks, and (C) Combi-medium: CnT-20 for the first week and then switched to SHEM+10% FBS for the second week. Cultures were evaluated by phase contrast, SEM and Transmission Electron Microscopy (TEM).

 

Results

 

SEM revealed a patterned surface of the decellularized scaffold (Fig 1). Phase contrast microscopy showed primary CECs cultured on the scaffolds displayed a homogeneous epithelial morphology (Fig 1). Immunofluorescence microscopy of cultured whole mounts showed positive staining for CK3/12, CK14, p63, ABCG2 and FLT4. In the optimization experiments, TEM of primary CECs cultured on scaffolds, following culture protocol A showed 3 to 4 layers of cellular stratification with CECs in several stages of differentiation. Cellular components and tight junctions were present. On SEM, the presence of numerous microvilli and cellular margins were detected. Culture protocol B showed very few CECs adhering to the scaffold, and a lack of desmosomes. Culture protocol C showed cellular stratification, clear desmosomes, but numerous large intercellular spaces.

 

Conclusions

 

We have been able to show that the decellularized fish scale derived scaffold is a biocompatible substrate with the potential for ophthalmic use as a bio-artificial cornea and that it is optimally cultured in SHEM+10%FBS for a period of 2 weeks for complete corneal epithelialization.

 

 

A. SEM image of the scaffold. The characteristic patterned channelling of the surface is observed. B. Phase contrast image overview of CEC growing within the patterned canals on the surface of the scaffold. ABCG2-FITC is shown in green and nuclei are stained with DAPI (blue).

 

Keywords: 482 cornea: epithelium • 599 microscopy: light/fluorescence/immunohistochemistry • 519 extracellular matrix